Dengue fever is one of most threatening mosquito-borne viral diseases in humans. It is caused by four closely related virus serotypes (DEN1, DEN2, DEN3 and DEN4) of the flavivirus genus within the Flaviviridae family. The dengue virus is transmitted by mosquitoes of the Aedes genus (particularly Aedes aegypti) and causes a febrile illness in tropical and sub-tropical regions. More than two billion inhabitants live in endemic regions and are at risk of dengue virus infection. According to the World Health Organization (WHO), the incidence of dengue infection increased 30-fold over the last 50 years (Dussart et al., Clin. Vaccine Immunol., 2006, 13:1185-1189), and is responsible for an estimated 50-100 million new infections annually, as well as 500,000 hospitalizations and 30,000 deaths, mostly in children.
Clinical signs of dengue infection usually appear five to eight days after infection and are characterized by undifferentiated fever (referred to as dengue fever), accompanied by severe headaches, lumbago, muscle and joint pain, and shivering. From the third to the fifth day of the febrile phase, a congestive maculopapular rash may appear (referred to as conventional dengue). In the majority of cases, disease usually subsides within a week. However, in the severe form, the infection can progress to hemorrhagic syndrome (referred to as dengue hemorrhagic fever) and fatal hypovolemic shock (referred to as dengue shock syndrome), which has a high mortality rate.
All four dengue serotypes are able to cause clinical symptoms of dengue infection, which provides a life-long immunity to the homologous dengue serotype, but does not protect against heterologous dengue serotype. Therefore, any person in an endemic area is susceptible to four infections during their lifetime. Secondary infection with a heterologous serotype may lead to more severe manifestations of disease, probably due to complement activation by antigen-antibody complexes (Young et al., J. Clin. Microbiol., 2000, 38:1053-1057; Xu et al., J. Clin. Microbiol., 2006, 44:2872-2878).
During the replication of dengue virus, a non-structural glycoprotein, NS1, associates with the membrane on the cell surface and is released into the circulation as early as 1 day post-onset of symptoms (Xu et al., 2006, Ibid). The early expression of the NS1 protein makes it a good diagnostic target for an early dengue infection as antibody-based diagnostic assays are capable of detecting dengue-specific antibody production later in the course of the infection (7-10 days post infection).
The mature dengue NS1 protein contains 352 amino acid residues in a base polypeptide of ˜40 kDa, with glycosylation increasing the apparent mass of the protein on SDS-PAGE (Flamand et al., J. Virol., 1999, 73:6104-6110). The protein includes twelve invariant cysteine residues and two N-glycosylation sites (at N130 and N207) conserved among all flavivirus NS1 proteins, indicating their importance to the structure and function of the protein.
NS1 exists in both intracellular and extracellular forms. Immature NS1 exists as a hydrophilic monomer in the endoplasmic reticulum lumen, and is rapidly processed into a stable hydrophobic non-covalent homodimer, with the subunits interacting via their carboxyl termini (Flamand et al., 1999, Ibid). Upon dimerization, NS1 becomes associated with intracellular membrane components. The ability to form intracellular dimers appears to be particularly important for trafficking and secretion from the cell. In mammalian cells, NS1 is secreted from infected cells into the extracellular milieu, either as a soluble protein, which may be present in a higher oligomeric form than a dimer, or in association with microparticles but not with virions. Studies have identified NS1 soluble tetramers and soluble, detergent-labile, hexamers (Wallis et al., J. Biol. Chem., 2004, 279: 20729-20741; and Flamand et al., 1999, Ibid). It has been postulated that the glycosylation status of NS1 determines the oligomeric distribution of secreted NS1. Even though the role of NS1 glycoprotein is not clearly defined, some studies have indicated that intracellular NS1 glycoprotein may be indirectly involved in viral replication, with extracellular NS1 glycoprotein being involved in the formation of immunogenic complexes and triggering complement mediated immune response, resulting in a more severe form of illness.
Currently, there is no commercially available vaccine for dengue virus. In the absence of immunization, the monitoring of dengue virus outbreaks and serological mapping of new outbreaks become critically important to the control and containment of infection. As clinical manifestations for dengue virus infections are quite unspecific, it is difficult to affirm diagnosis without laboratory testing. Programs have been set up by WHO to actively monitor vector insects and cases of fever, as well as to perform serological and virological screening of individuals suspected of being infected with dengue virus. Thus, the development of diagnostic assays for dengue infection is critically important.
Early diagnosis is essential for proper timely treatment of the patient. The currently available tests for dengue include RT-PCR for viral RNA and immunologic tests for dengue-specific antibody or viral proteins. However, many of these tests have significant disadvantages. For example, RT-PCR for viral RNA requires expensive laboratory equipment and trained personnel, which makes it hard to use on a large scale or in rural areas. Some dengue-specific enzyme linked immunosorbent assays (ELISAs) can detect IgM or IgG that appear later during the course of infection, however diagnosis as early as day two of infection is preferable (Alcon et al., J. Clin. Microbiol., 2002, 40:376-381).
A comparative analysis of four diagnostic methods for dengue infection, namely virus isolation, viral RNA detection, dengue specific IgM detection and NS1 antigen detection, revealed that NS1 antigen detection had the highest sensitivity rate compared to the other three methods (Kumarasamy et al., Singapore Med. J., 2007, 48:669-673). Several immunological tests employing specific peptides (including NS1) derived from dengue virus have been proposed. U.S. Pat. Nos. 7,282,341, 6,870,032, 5,824,506, 6,682,883, 6,190,859 and PCT Patent Publication WO99/009414 describe methods of using peptides as a diagnostic tool for determining the presence of dengue virus.
Alcon et al. (J. Clin. Microbiol., 2002, 40:376-381) have described an ELISA for NS1 detection and demonstrated that NS1 is present at high levels in patient sera during primary and secondary infection. NS1 is detectable during the whole clinical phase of illness and can be detected in the first few days of infection (as early as the first day of fever). Falconar and Young have described the production of dimer-specific and dengue virus group cross-reactive mouse monoclonal antibodies to dengue 2 virus NS1 (J. Gen. Virol., 1991, 72:961-965), and the use of certain of these antibodies in an ELISA for NS1 has been described (Young et al., J. Clin. Microbiol., 2000, 38:1053-1057). High levels of NS1 were found in acute phase sera, but not in convalescent phase sera, from some of the patients with serologically confirmed dengue 2 virus secondary infection.
U.S. Pat. No. 6,870,032 describes a method for detecting NS1 protein in the hexameric form, and the selection of antibodies specific for NS1 protein in hexameric form, together with the use of such antibodies in the early detection of flavivirus infection. All these studies demonstrate that antibodies directed to different epitopes of NS1 and NS1 oligomeric forms, as well as NS1-immune complexes, may play different roles in the diagnosis of dengue virus infection.